Herpes viruses comprise a large family of complex, double-stranded DNA viruses, a number of which are serious human pathogens. Herpes DNA replication requires a group of virally encoded proteins, and is the target of several antiviral drugs. However, the mechanism of Herpes DNA replication is remarkably complex and not well understood. The long term goal of these studies is to understand the mechanism of Herpes DNA replication at the level of individual enzymes. The studies in this proposal will focus on one of the key reactions in DNA replication, the initiation of new strands of DNA by the DNA primase/helicase complex and subsequent transfer of the primer to the Herpes DNA polymerase. The specific aims of this proposal are: 1. Obtain a detailed understanding of the mechanism of primer synthesis within the context of the primase/helicase complex. Three aspects of primase activity will be examined; how primase recognizes a potential primer synthesis site, the mechanistic coupling between the primase and helicase activities, and the fate of primase synthesized primers. 2. Develop a detailed understanding of the fidelity of primase. The mechanism(s) by which Herpes primase misincorporates NTPs, as well as the frequency and spectrum of misincorporation will be determined. 3. Elucidate the mechanism of primase-coupled DNA polymerase activity. How primase-synthesized primers are transferred between the primase and polymerase as well as the effects of the polymerase on primase activity will be ascertained. 4. Determine how primase interacts with both the base and sugar of the incoming NTP by examining the interaction of primase with NTP analogs. The data from these studies will be combined and used to direct the synthesis of novel and highly specific inhibitors of primase. To accomplish these aims, a variety of steady-state kinetic approaches will be employed. These approaches will be augmented with studies using photoactivateable crosslinking reagents along with a selection-based methodology to provide insights into the interaction of the enzyme with substrates and products. Additionally, a number of novel nucleotides will be synthesized to provide a thorough understanding of how the enzyme interacts with the incoming NTP.